Wortmannin is a fungal metabolite found to be a potent catalytic inhibitor of phosphatidylinositol-3(OH)-kinase (PI3K) and TOR kinase function within signal transduction pathways. (Norman, Bryan H., et al. (1996) “Studies on the Mechanism of the Phosphatidylinositol 3-Kinase Inhibition by Wortmannin and Related Analogs,” J. Med. Chem., 39, 1106-111 and Creemer, Lawrence C. (1996) “Synthesis and in Vitro Evaluation of New Wortmannin Esters: Potent Inhibitors of Phosphatidylinositol 3-Kinase,” J. Med. Chem., 39, 5021-5024).
Class-1a PI3K (referred to as PI3K) is a heterodimeric enzyme comprised of the p85 regulatory and p110 catalytic subunits. In response to growth factor receptor stimulation, PI3K catalyzes the production of the lipid second messenger phosphatidylinositol-3,4,5-triphosphate (PIP3) at the cell membrane. PIP3 in turn contributes to the activation of a wide range of downstream cellular substrates. The most critical signaling mediators downstream of PI3K include the serine/threonine kinase AKT and the mammalian target of rapamycin (mTOR). AKT confers a dominant survival signal and promotes proliferation via direct phosphorylation of multiple cell death/apoptosis proteins and cell cycle factors. mTOR is a central regulator of cell growth via controlling cellular protein translation. Thus, the PI3K/AKT/TOR pathway is critical for cell proliferation, growth, survival and angiogenesis.
In human cancer, deregulation in the PI3K/AKT/TOR pathway is among the most frequent events occurring in all major human tumors. Genetic loss of the tumor suppressor gene PTEN, a PIP3 phosphatase and a negative regulator of the PI3K signaling, is estimated to occur in 30-50% of all human cancers including lung, prostate, breast, brain, renal, melanoma, ovarian, endometrium, thyroid and lymphoid. In addition, constitutive elevation of PI3K expression has been associated with lung, ovarian and pancreatic cancers. Finally, cell surface oncogenes such as Her-2, EGFR and Ras cause constitutive PI3K signaling in breast, prostate, colon and lung tumors. These clinical data provide a strong rationale for exploring PI3K inhibitors as novel anticancer agents. (Cantley, L. and Neel, B. (1999) “New Insights into Tumor Suppression: PTEN Suppresses Tumor Formation by Restraining the Phosphoinositide 3-kinase/AKT pathway,” Proc. Natl. Acad. Sci. USA, 96, 4240-4245). PI 3 kinase and TOR kinase have been shown to be active in cancer (Vivanco, I. and Sawyer, C. (2002) “The phosphatidylinositol 3-kinase-AKT Pathway in Human Cancer,” Nature Reviews Cancer, 2, 489-501), iscaemic heart disease and restenosis (Shiojima, I. And Walsh, K. (2002) “Role of Akt Signaling in Vascular Homeostasis and Angiogenesis,” Circulation Research, 90, 1243-1250 and Ruygrok P., et al. (2003) “Rapamycin in Cardiovascular Medicine,” Intern Med J., 33, 103-109), inflammation (Wymann, M., et al. (2003) “Phosphoinostide 3-kinase gamma: A Key Modulator in Inflammation and Allergy,” Biochem Soc Trans, 31, 275-280 and Kwak, Yong-Geun, et al. (April 2003) “Involvement of PTEN in airway hyperresponsiveness and inflammation in bronchial asthma,” The Journal of Clinical Investigation, 111:7, 1083-1092), platelet aggregation (Watanabe, N., et al. (March 2003) “Functional Phenotype of Phosphoinositide 3-kinase p85 (alpha) Null Platelets Characterized by an Impaired Response to GP VI Stimulation,” Blood (epub)), sclerosis (Kenerson, H., et al. (2002) “Activated Mammalian Target of Rapamycin in the Pathogenesis of Tuberous Sclerosis Complex Renal Tumors,” Cancer Res., 62, 5645-5650), respiratory disorders (Kitaura, J., et al. (2000) “AKT-dependent Cytokine Production in Mast Cells,” J. Exp. Med., 192, 729-739 and Stewart A. (2001) “Airway Wall Remodeling and Hyper-responsiveness: Modeling Remodeling in vitro and in vivo,” Pulm Pharmacol Ther, 14, 255-265), HIV (Francois, F. and Klotman, M. “Phosphatidylinositol 3-kinase Regulates Human Immunodeficiency Virus Type-1 Replication Following Viral Entry in Primary CD4(+) T Lymphocytes and Macrophages,” J. Virol., 77, 2539-2549), and bone resorption (Pilkington, M., et al. (1998) “Wortmannin Inhibits Spreading and Chemotaxis of Rat Osteoclasts in vitro,” J Bone Miner Res, 13, 688-694).
PI3K exists as a tightly associated heterodimer of an 85 kDa regulatory subunit and 110 kDa catalytic subunit, and is found in cellular complexes with almost all ligand-activated growth factor receptors and oncogene protein tyrosine kinases (Cantley, L. C., et al., Cell, 64:281-302 (1991)). The 85 kDa regulatory subunit apparently acts as an adaptor of PI3K to interact with growth factor receptors and tyrosine phosphorylated proteins (Margolis, C., Cell Growth Differ., 3:73-80 (1992)).
Although PI3K appears to be an important enzyme in signal transduction, with particular implications relative to mitogenesis and malignant transformation of cells, only a limited number of water-soluble drug-polymer conjugates have been identified as having inhibitory activity against PI3K (see, e.g., Matter, W. F., et al., Biochem. Biophys, Res. Commun., 186:624-631 (1992)). Contrary to the selective PI3K activity of the water-soluble drug-polymer conjugates used in the methods of the present invention, the bioflavinoid water-soluble drug-polymer conjugates used by Matter, et al., particularly quercetin and certain analogs thereof, inhibit PI3K and other kinases such as protein kinase C and PI 4-kinase (Id.).
U.S. Pat. No. 5,378,725, issued Jan. 3, 1995, provided a method for inhibiting PI3K in mammals using wortmannin or one of certain analogs thereof. One of the disadvantages of wortmannin is its toxicity to living creatures. Even in low dosages, wortmannin in pure form is often systemically dose limiting to laboratory animals.
The biosynthetic production of wortmannin is well known in the art and the derivatives are synthesized from wortmannin. (Dewald, Beatrice, et al. (1988) “Two Transduction Sequences Are Necessary for Neutrophil Activation by Receptor Agonists,” The Journal of Biological Chemistry, Vol. 263, Issue of November 5, pp 16179-16184; Norman, Bryan H., et al. (1996) “Studies on the Mechanism of Phosphatidylinositol 3-Kinase Inhibition by Wortmannin and Related Analogs,” J. Med. Chem., 39, pp 1106-1111; Varticovski, L., et al. (2001) “Water-soluble HPMA copolymer-wortmannin conjugate retains phosphoinositide 3-kinase inhibitory activity in vitro and in vivo,” Journal of Controlled Release, 74, pp 275-281), all hereby incorporated by reference.
A wortmannin derivative, 17β-Hydroxywortmannin prepared from the reduction of wortmannin with diborane, showed a 10-fold increase in activity relative to wortmannin and pushed the PI3K IC50 into the subnanomolar range, with an IC50 of 0.50 nM. However, antitumor activity of 17β-Hydroxywortmannin in the C3H mammary model showed no inhibition at a dose of 0.5 (mg/kg) and toxicity at a dose of 1.0 mg/kg. These findings lead the authors to conclude, “nucleophilic addition to the electrophilic C-21 position of wortmannin and related analogs is required for inhibitor potency and antitumor activity. Unfortunately, this mechanism appears to be linked to the observed toxicity” (Norman, Bryan H., et al. (1996) “Studies on the Mechanism of Phosphatidylinositol 3-Kinase Inhibition by Wortmannin and Related Analogs,” J. Med. Chem., 39, 1106-1111, 1109-1110).
Wortmannin derivatives acetylated at the C-17 hydroxyl group showed a dramatic loss in activity leading the authors to conclude, “the active site cannot accommodate liphophilicity or steric bulk at C-17” (Creemer, Lawrence C., et al. (1996) “Synthesis and in Vitro Evaluation of New Wortmannin Esters: Potent Inhibitors of Phosphatidylinositol 3-Kinase,” J. Med. Chem., 39, 5021-5024, 5022). This conclusion is consistant with the X-ray crystallographic structure of PI3K bound to wortmannin subsequently elucidated (Walker, Edward H., et. al (2000) “Structural Determinants of Phosphoinositide 3-Kinase Inhibition by Wortmannin, LY294002, Quercetin, Myricetin, and Staurosporine,” Molecular Cell, 6(4), 909-919).
Other wortmannin derivatives are opened at C-20. By reacting wortmannin with nucleophiles at the C-20 position, the furan ring is opened. Such ring-opened compounds demonstrate a range of biological activities (Wipf, Peter, et al. (2004) “Synthesis and biological evaluation of synthetic viridins derived from C(20)-heteroalkylation of the steroidal PI-3-kinase inhibitor wortmannin,” Org. Biomol. Chem., 2, 1911-1920). See also U.S. 2003/0109572 to Powis.
Attaching poly(ethyleneglycol) (PEG) has been successfully employed in medicinal chemistry to improve the aqueous solubility and administration of drugs. (Id.) However, covalently attaching PEG does not necessarily offer improvement in water solubility and availability of the drug to which it is attached (Bebbington, David, et al. (2002) “Prodrug and Covalent Linker Strategies for the Solubilization of Dual-Action Antioxidants/Iron Chelators,” Bioorganic & Medicinal Chemistry Letters, 12, 3297-3300, 3299) and (Feng, Xia, et al. (2002) “Synthesis and Evaluation of Water-Soluble Paclitaxel Prodrugs,” Bioorganic & Medicinal Chemistry Letters, 12, 3301-3303, 3302).
In an overview of PEG drugs, no low molecular weight (<20,000) PEG small molecule drug conjugates, prepared over a 20-year period, have led to a clinically approved product (Greenwald, R. B. (2001) “PEG drugs: an overview,” Journal of Controlled Release, 74, pp 159-171, abstract). In fact only a few small organic molecule anticancer agents have been conjugated to PEG with permanent bonds, and those did not lead to clinically superior water-soluble drug-polymer conjugates (Greenwald, R. B., et al. (2003) “Effective Drug Delivery by PEGylated Drug Conjugates,” Advanced Drug Delivery Reviews, 55, pp 217-250, 220). Using PEG-CPT, lethality was demonstrated to be approximately 50%, 10% and 0% for the PEG-CPT 40,000, 20,000 and 8,000 constructs. Ostensibly, employing polymer Mw 5000 to conjugate drugs gave rapidly excreted species that would have little or no effect in vivo (Id., 225). That is not to say the attachment of PEG 40,000 with its ability to accumulate in tumors will automatically permit drugs to have greater antitumor activity (Id., 235).
There is a need for wortmannin analogs with improved antitumor activity and/or low toxicity. Compounds of the present invention fulfill this need.